摘要
目的观察不同类型DNA聚合酶β(DNA Polymerase β,DNA pol β)在小鼠成纤维细胞(NIH3T3细胞)的表达及细胞增殖特性的变化。方法用脂质体转染的方法,将野生型(wild type,wt)和食管癌缺失突变型(delete mutional type,dmt)的polβ重组荧光载体pEGFP-C3-polβ转染NIH3T3细胞,建立稳定表达polβ的细胞系,通过荧光倒置显微镜观察其定位,四甲基偶氮噻唑蓝(MTT)比色法绘制生长曲线,流式细胞仪测定其细胞周期。结果转染2种类型polβ的NIH3T3细胞株中,均有外源基因mRNA水平和蛋白水平的表达;荧光倒置显微镜结果显示,野生型的以细胞胞核为主,缺失突变型的polβ表达以细胞胞浆为主,从第3d开始,缺失突变型的细胞生长速度较野生型和对照组明显增快(P<0.05);转染缺失突变型NIH3T3细胞的S期细胞比例为68.29%,明显高于野生型的57.18%、对照组的58.12%和未转染NIH3T3细胞的57.35%(P<0.05)。结论外源野生型polβ基因表达后对NIH3T3细胞的增殖速度无明显影响,而缺失突变型则使其增殖速度加快。
Objective To observe the proliferation characteristics of NIH3T3 cell line under expression of different type DNA polymerase β （DNA polβ）.Methods The wild polβ and delete mutational polβ of esophageal cancer cells were transfected into NIH3T3 cell line by lipofectamine method.The stable expression polβ NIH3T3 line was constructed.The location of polβ gene-encoded protein was observed with fluorescent microscope.The growth curve was drawn with MTT method and the cell cycle was examined with flow cytometer.Results It was proved that two polβ genes were transfected into the NIH3T3 cell line successfully.The location of wild type polβ protein was mostly inside the nuclear,but the delete mutational polβ protein was distributed in the whole cell.The grow speed of NIH3T3-wild type（wt） polβ was not obviously different compared with control cells and NIH3T3-EGFP（P0.05）.But the grow speed of NIH3T3-delete mutational type（dmt） tpolβ was faster obviously（P0.05）.Furthermore,the cell account of S period of NIH3T3-dmt polβ was obviously increased（P0.05）,but that of NIH3T3-wt polβ showed no obvious change（P0.05）.Conclusion Expression of exo-genous wild polβ is no obvious effect on the proliferation of NIH3T3 cell and the expression of exogenous delete mutational polβ can make NIH3T3 cells proliferate.The result has great significance to further dicuss the pathogenesis of esophageal cancer and provides a strong basis for esophageal cancer biotherapy.
出处
《中国公共卫生》
CAS
CSCD
北大核心
2010年第1期106-108,共3页
Chinese Journal of Public Health
基金
国家自然科学基金(30471952)
